PROJECT SUMMARY/ABSTRACT Current approaches in repairing craniomaxillofacial (CMF) defects possess several limitations and the reconstruction of CMF defects seamlessly is highly challenging, as precise layer-by-layer stacking of multiple tissue compartments is not trivial. Such compartmentalization necessitates the precision and effective use of stem cells and differentiation factors, and differentiating stem cells into multiple lineages is crucial in order to recapitulate the native tissue anatomy. With the advances in three-dimensional (3D) bioprinting, reconstruction of composite tissues in situ for CMF repair has recently become feasible as 3D bioprinting enables complex tissue heterogeneity in an anatomically accurate and cosmetically appealing manner. Intraoperative bioprinting, which can be defined as bioprinting directly into the defect area for repairing injuries in a surgery setting, is a highly effective process for CMF reconstruction, where the defect information can be rapidly acquired with minimum manual interventions, enabling accurate personalized reconstructions immediately after characterization of the defect. In this project, we hypothesize that intraoperatively bioprinted multi-layer composite tissues loaded with differentiation factors including microRNA(miRNA)-transfected human progenitor cells and adipose-derived extracellular matrix components (adECM) induce compartmentalization of soft and hard tissues that recapitulates CMF tissue anatomy on an athymic rat model. In order to test our hypothesis, Specific Aim I will use intraoperative bone tissue bioprinting to reveal the impact of miR-148b- transfected human adipose-derived stem cells (ADSCs) at respective dosages on bone tissue regeneration. In Specific Aim II, we will intraoperatively bioprint multi-layer skin tissues, including adipose and dermis layers, in order to explore the impact of localized delivery of human ADSCs and adECM components at different dosages and concentrations on skin tissue regeneration, respectively. Particularly, we will observe if ADSCs differentiate into adipocytes and also understand the impact of the presence of adipose layer on dermis regeneration. In Specific Aim III, we will intraoperatively bioprint three-layer composite tissues, including cranium, adipose and dermis layers, in order to understand the role of a vascularization on soft and hard tissue regeneration. In addition, we will explore the role of miR-210 in vascularization. In this regard, we have formed a complementary collaboration that merges essential domain knowledge in bioprinting, regenerative medicine, CMF surgery, plastic surgery, gene therapy, gene delivery, bone mechanics, and bone and skin biology with the depth necessary to propel the proposed work towards meaningful advances that would otherwise not be possible. Successful completion of the proposed work is anticipated to give rise to an advanced bioprinting technology revealing the complex interactions between stratified layers of engineered tissues in an immunodeficient rodent model and thereby provide a novel understanding of how localized delivery of differentiation factors impacts craniomaxillofacial reconstruction.